Membrane transporter proteins may play a role both in the pharmacokinetics of several drugs (when expressed in normal tissues) and/or resistance to several anticancer agents (when expressed in cancers). Several of these proteins belong to the ABC (ATP Binding Cassette) protein superfamily, including, P-glycoprotein (Pgp, ABCB1), the Multidrug Resistance Protein (MRP1, ABCC1), the Mitoxantrone Resistance Protein (MXR, ABCG2), the canalicular Multispecific Organic Anion Transporter (cMOAT, ABCC2), the Bile Salt Export Pump (BSEP, ABCB11). Other transporter proteins, such the Organic Cation transporters (OCT1 and OCT2) and the Organic Anionic Transporter (OAT) do not belong to the ABC superfamily. These proteins seem to share several substrate drugs including, possibly, some anticancer agents. The partial sharing of anticancer and non-anticancer substrates between different transporters may explain some of the side effects of drugs used to inhibit the function of specific transporters. For example, Cyclosporin A has been tested clinically as an inhibitor of Pgp-caused multidrug resistance. One of its side effects is jaundice, which might be caused by its interference of the transport of conjugated bilirubin by cMOAT. So, the clinical efficacy of transporter inhibitors (whether used to reverse anticancer drug resistance, or to modulate pharmacokinetics) may be improved by optimizing their transporter selectivity. Pgp is the transporter that has been most thoroughly studied. Pgp effluxes its substrates from cells by transporting them from the cell membrane's inner to the outer leaflet or to the extracellular space. Central to the biology of Pgp is its ability to bind a wide array of diverse substrates and inhibitors, suggesting the possible existence of multiple binding sites. The lack of definition of these sites and the unavailability of a crystal structure for Pgp have so far hindered a rational design of drugs targeting Pgp function. We propose that affinity chromatography can be used as a means to characterize the binding sites and transport cycle of the different transporters and, ultimately, to define effective and protein-selective pharmacophores for the pharmacological inhibition of transporter function. We are presently characterizing and optimizing affinity chromatography models of Pgp. This process will also provide a prototype for the modeling of other ABC and non-ABC transporters. Pgp immobilization into the stationary phase of chromatographic columns is obtained in three steps: first, Pgp is extracted by detergent from the membranes of Pgp positive cells (MDA435/LCC6MDR1 human breast cancer cells); second, Pgp is reconstituted by dialysis into Immobilized Artificial Membranes on silica particles (IAM beads); finally, the Pgp-IAM beads are packed into different kinds of chromatographic columns (so, we have used either Amersham's HR5/2 glass chromatographic columns or peek tubing). Negative control columns are obtained using membranes form parental Pgp-negative LCC6/MDA435 cells. Frontal and zonal chromatography has confirmed an increased retention of Pgp substrates, which is specifically evident in Pgp' positive columns. Substrate displacement studies in frontal chromatography allow to calculate substrate affinity and number of binding sites. Comparison of affinity chromatography and classical filtration binding assays, shows a partial overlapping in the definition of the affinity of different substrates. Similar results were obtained for vinblastine and doxorubicin, but not for verapamil and cyclosporin A, suggesting that the IAM model may not be completely representative of binding to Pgp in its native membrane environment, and that alternative methods of immobilization should be evaluated. Affinity chromatography can be used to evaluate reciprocal displacement by different substrates, and so also to evaluate whether two substrates bind to the same or to different sites. The results of our evaluations so far provide suggestions about the nature of interactions between several substrates, in the absence of added ATP. Verapamil binds to multiple sites on Pgp, at least one of which is shared with vinblastine. Cyclosporin A does not bind to Pgp, in the absence of ATP and/or other substrates, but its binding is elicited by vinblastine. Cyclosporin A, however, displaces vinblastine, suggesting an interaction which is neither comeotitve nor allosteric as it is not reciprocal. (+)Mefloquine and (-)mefloquine interact with cyclosprin A and vinblastine in a manner which is enantio-selective for the former, but not for the latter. More extensive evaluations of substrate displacement by frontal chromatography and by non-linear zonal chromatography analysis will allow to complete a functional definition of binding sites by identifying which substrates share the same sites. It has to be noted that it is possible that these evaluations are not optimal in the absence of ATP, as the conformation of binding sites may be quite different when ATP is bound to Pgp and before ATP hydrolysis. The latter conformation may be more relevant to the definition of effective pharmacophores. We have observed that addition of ATP changes the affinity for its substrates: affinity for vinblastine and verapamil is decreased, while that for cyclosporin A is increased. Binding to Pgp in the presence of ATP is bound to represent the "average" of Pgp's affinities for its substrates as it goes through the different steps of the transport cycle, including the steps that immediately precede and follow the catalysis of ATP to ADP and inorganic phosphate. It is to be expected that affinity for effective substrates will be decreased in the post-catalytic stage, when the substrate has to be released to the extracellular side of the membrane. As in the case of filtration binding assays, affinity chromatography can be used to model the different stages of Pgp's transport cycle. For example, use of non-hydrolysable ATP analogs will allow to model Pgp in the "pre-catalytic" stage, and pre-treatment of the Pgp column with vanadate and ATP in the presence of a substrate will "freeze" it in the catalytic stage. In preliminary studies, we have used zonal affinity chromatography to evaluate vinblastine binding to a "vanadate-trapped" Pgp-column. The results have confirmed inhibition of vinblastine binding to post-catalytic Pgp. These evaluations will be extended to different substrates and to different stages of the transport cycle. In summary, we have obtained and are characterizing an affinity chromatography model for the functional evaluation of Pgp binding sites and transport cycle. We will try to further optimize our affinity chromatography models and we may, for example, evaluate chromatographic columns where different approaches are used to immobilize Pgp in its native membrane. We plan then to proceed to full functional characterization of Pgp's binding sites by identifying those substrates that share the same site, at different stages of the transport cycle. Structure-affinity relationships of site-sharing substrates will be used to define pharmacophores. Other ABC and non-ABC transporters (such as MRP1 and OCT1) will be modeled and comparison of results will be used to define transporter-selective pharmacophores.